The anion exchange channel (band 3) is a 95 k transmembrane protein in the human erythrocyte which facilitates the physiologically important exchange of HCO3 for Cl. The cytoplasmic domain of band 3 provides a membrane binding site for glycolytic enzymes and hemoglobin as well as attachment site for bridging the membrane skeleton to the lipid bilayer. These latter functions suggest that band 3 acts as an organizing center for protein-protein interactions which are crucial to the maintenance of the biconcave shape and to the unusual viscoelastic properties of the erythrocyte. The long term goals of the proposed research are to characterize structural features of band 3 which are relevant to its function as an anion transport protein and to provide an increased understanding of its organizing functions through elucidation of dynamic properties of specific membrane skeleton-band 3 interactions. Two reactively bifunctional spin label reagents, bis)sulfosuccinimidyl)-4-doxylpimelate (BSSDP) and bis(sulfosuccinimidyl)-5-doxylazelate (BSSDA), have been developed which allow selective cross-linking of band 3 at residues in its extracellular anion binding domain or at an extracellular monomer-monomer contact surface in intact erythrocytes. Protein sequencing will now be employed to define the amino acid residues on band 3 which are cross-linked by the spin label probes at both sites. The spatial arrangement of these functionally and structurally important sites will be investigated by electron paramagnetic resonance (EPR) spectroscopy by analyzing line shapes for probe-probe interactions. These same spin labels will be employed in saturation transfer EPR (ST-EPR) measurements for quantitating the rotational motion of band 3 in intact erythrocytes and ghost membrane preparations under a variety of physiological conditions. The aim of these studies is to elucidate dynamic properties of specific interactions between membrane skeletal proteins and band 3 using rotational motion as a probe for the nature and extent of the interactions. These studies will also be extended to include erythrocytes exhibiting abnormal shape and fragility resulting in hemolytic anemia due to altered interactions between members of the membrane skeleton and/or its attachment to band 3. Complementary ST-EPR studies will be carried out on maleimide spin labeled ankyrin and protein 4.2 bound to erythrocyte ghost membranes in oder to define the flexibility of contact between the cytoplasmic domain of band 3 and the underlying membrane skeleton.